ABSTRACT Recent preclinical studies have shown that different subsets of both helper CD4+ T helper (Th) cells and CD8+ T cytotoxic (Tc) cells hold promise for clinical use in adoptive cell therapy (ACT) protocols. Importantly, T helper cell subsets with the ability to secrete IL-17 (Th17) have been shown to possess stem cell like phenotype that attributes to their long-term persistence and leads to improved tumor control tumors as compared to the Th1 subsets (that secrete IFN?, IL2, TNF?). However, contrary to these observations there are reports that Tc1 cells exhibit improved tumor control as compared to Tc17 cells. These differences in T cell subsets response to control tumors is compounded by the fact that in the suppressive tumor microenvironment a large fraction of tumor reactive T cells acquire FoxP3+ regulatory phenotype, become dysfunctional or undergo cell death leading to tumor reversion. Thus, ex vivo programming conditions that can render T cells a stable phenotype, which not only controls primary tumors but also results in the formation of anti-tumor memory will be of immense importance for ACT. We have recently established that programming conditions that bring together ?anti-tumor effector function? of Th1 cells and ?stemness? of Th17 cells lead to a superior hybrid Th1/17 cells exhibiting long-term tumor control. Importantly, these ex vivo programming conditions also generate highly effective hybrid Tc1/Tc17 cells and render human tumor infiltrating lymphocytes (TILs) with increased cytokine secreting ability. Importantly, the hybrid T cells exhibited higher levels of nicotinamide adenine dinucleotide (NAD+), a cofactor that serves as substrate for Sirtuins and regulates multiple metabolic and epigenetic molecules. We hypothesize that the robust long-term tumor control was observed with hybrid Th1/17 cells was mediated by an overall rejuvenated T cell phenotype due to high NAD+ that influenced a combination of events including post-translational modifications, and epigenetic stability that led to metabolically fit phenotype. Given the crucial role of NAD+ in a variety of biological processes including energy metabolism, aging, calcium homeostasis, and epigenome, we propose the following aims to test the above hypothesis: Aim 1) To determine if metabolic signature and anti-tumor phenotype of the cytokine subsets in hybrid T1/17 cells could be translated to program tumor infiltrating lymphocytes; Aim 2) To determine if antibody mediated combinatorial inhibition of CD38 with PD1 antibody leads to robust anti-tumor response in different preclinical in vivo and xenograft models; and Aim 3) To determine how NAD+ level contributes to metabolo-epigenetic programming that preserves robust anti-tumor response in T1/17 hybrid and CD38-KO T cells. We believe that this proposal to determine the central mechanism that results in superior anti-tumor response by hybrid T1/17 cells will be important to adapt these ex vivo programming conditions for immediate translational use in adoptive T-cell immunotherapy.